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Trends Biotechnol., Nov 06 2025, S0167-7799(25)00416-0 | https://doi.org/10.1016/j.tibtech.2025.10.009 Integrated Tn-seq and MAGE-assisted rapid genome engineering targeting in Escherichia coli

Jaeseong Hwang^1,2,15, Yong Hee Han ^3,4,5,15, Ina Bang ⁶, Joon Young Park⁶, Donghyuk Kim ⁶, Junyeong Sung ², Sang Woo Seo ^5,7,8,9,10, Sungho Jang ^11,12,13, Gyoo Yeol Jung^2,14,16

¹Advanced Convergence Research Division, World Institute of Kimchi, 86 Kimchi-ro, Nam-gu, Gwangju 61755, Republic of Korea
²Department of Chemical Engineering, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-gu, Pohang, Gyeongbuk, Republic of Korea
³School of Biological Sciences and Technology, Chonnam National University, 77 Yongbong-ro, Gwangju 61186, Republic of Korea
⁴Institute of Systems Biology and Life Science Informatics, Chonnam National University, 77 Yongbong-ro, Gwangju 61186, Republic of Korea
⁵Interdisciplinary Program in Bioengineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
⁶School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea
⁷School of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
⁸Institute of Chemical Processes, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
⁹Bio-MAX Institute, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
¹⁰Institute of Bio Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
¹¹Department of Bioengineering and Nano-Bioengineering, Incheon National University, 119 Academy-ro, Yeonsu-gu, Incheon 22012, Republic of Korea
¹²Division of Bioengineering, College of Life Sciences and Bioengineering, Incheon National University, 119 Academy-ro, Yeonsu-gu, Incheon 22012, Republic of Korea
¹³Research Center for Bio Materials and Process Development, Incheon National University, 119 Academy-ro, Yeonsu-gu, Incheon 22012, Republic of Korea
¹⁴Division of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, 77 Cheongam-ro, Nam-gu, Pohang, Gyeongbuk 37673, Republic of Korea
¹⁵These authors contributed equally
¹⁶Lead contact

Correspondence: Sang Woo Seo, Sungho Jang, Gyoo Yeol Jung

Abstract

Improving microbial strains is essential for the economic feasibility of bio-based chemical production; however, the intricate nature of metabolic networks and gene interactions makes identifying effective genetic engineering targets challenging. We developed iTARGET, an integrated approach combining in situ transposon mutagenesis, biosensor-guided selection, and multiplex automated genome engineering (MAGE) to identify novel and synergistic genetic targets that are challenging to predict through rational design. In the first phase, in situ transposon mutagenesis generated genetic diversity within a single batch culture, allowing biosensor-driven enrichment of high-producing mutants. Transposon sequencing (Tn-seq) was then performed to identify critical genomic targets. In the second phase, MAGE enabled the creation of combinatorial knockout (KO) libraries, and high-throughput screening revealed synergistic gene interactions. Applying iTARGET to naringenin (NRN) production enriched high-producing mutants, achieving a population-level titer 1.7-fold higher than that in the control. Next-generation sequencing identified nine unpredictable genetic targets, achieving a 2.3-fold titer increase with single KOs. Further combinatorial KOs revealed synergistic effects, with a double-KO mutant producing a 2.8-fold improvement. By integrating mutagenesis and selection into a single batch, iTARGET accelerates the discovery of challenging genetic targets and enables the exploration of synergistic gene interactions through high-throughput identification of combinatorial KOs, enhancing bio-based chemical production.

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